The proposed research on the catalytic mechanism of papain, a model sulfhydryl protease, is an outcome of two previous years work focusing on the mechanism by which an imidazole function neighboring the active site sulfhydryl group could promote the acylation reaction between the sulfhydryl and polypeptide substrates (D.J. Creighton and David J. Schamp, FEBS Letters, 110, 313 (1980); D.J. Creighton et al., FEBS Letters, 319 (1980); A. Wandinger and D.J. Creighton, FEBS Letters, 116, 116 (1980). An active site thiolate-imidazolium ion pair appears to be the most reactive nucleophilic form of the enzyme based on kinetic solvent deuterium isotope effect studies. The tautomerization constant controlling the equilibrium distribution of the thiolate-imidazolium ion pair and its thiol-imidazole tautomer is estimated to be approximately 2 in H2O based on the effect of solvent D2O on selected spectral properties of the enzyme. In the proposed work, the tautomerization constant is to be independently determined by estimating the microscopic ionization constant of the active site imidazole when the sulfhydryl is protonated. This will be done from the pH-dependence of protein fluorescence using papain-S-CH3 as an analog of papain-SH. Additional studies are planned for examining the state of ionization of the active site sulfhydryl of streptococcal protease during the zymogen to enzyme transformation.